Dengue virus (DV) is a single-stranded RNA flavivirus that causes dengue fever, the most prevalent arthropod-borne viral illness in humans (NIAID Category A pathogen). Globally, the four serotypes of DV cause an estimated 100 million new cases of dengue fever and 250,000 cases of dengue hemorrhagic fever (DHF) per year, primarily in tropical and subtropical areas. Currently, no specific therapy is available for human use. Given its global burden, there is a pressing need for the development of safe therapeutics against DV. Recently, we defined a dominant neutralizing epitope on domain III of the envelope (E) protein o West Nile virus (WNV), a related flavivirus, and humanized one monoclonal antibody (Hu-E16) with clinical potential as a post-infection therapeutic. Hu-E16, which is now entering human clinical trials, binds to a conserved WNV-specific epitope, and neutralizes at a post-attachment step of infection. While in theory, antibody-based therapeutics could have analogous activity against DV, the risk for antibody-dependent enhancement of infection (ADE) with adverse outcome has limited this approach. For this proposal, we will use an existing academic-industry collaborative partnership to generate an antiviral biological product consisting of humanized monoclonal antibodies (mAbs) that potently neutralize all serotypes of DV without he risk of ADE. In Specific Aim 1, we will generate mouse mAbs against the E protein of all four DV serotypes and test their relative neutralizing potential in vitro and in vivo. In Specific Aim 2, we will identify recognition determinants on DV E proteins for the strongly inhibitory mAbs using high-throughput yeast surface display epitope mapping. Using this information, we will create DV variant strains that are resistant to mAb neutralization and test them for virulence. In Specific Aim 3, we will humanize candidate inhibitory mAbs that strongly block infection against each DV serotype. These will be tested for neutralizing activity in cell culture and in mice. In Specific Aim 4, we will engineer safe DV immunotherapeutics by altering the effector functions of candidate humanized mAbs. Cell lines producing IgG subclass variants will be developed that strongly neutralize yet abolish Fc receptor binding and prevent ADE. Overall, the generation of humanized mAbs against all four DV serotypes with no possibility of ADE will foster the development of potent and safe immunotherapeutics against DV infection.